Mining for gems in the fungal genome

Ever since penicillin, a byproduct of a fungal mold, was discovered in 1929, scientists have scrutinized fungi for other breakthrough drugs. As reported Jan. 20 in the Journal of Chemistry and Biology, a team led by a UW-Madison researcher has developed a new method that may speed the ongoing quest for medically useful compounds in fungi.

By manipulating a single fungal protein, the team, led by professor of plant pathology and medical microbiology Nancy Keller, pinpointed the genes responsible for creating dozens of secondary metabolites, a class of compounds that make good drug candidates. Already, analysis of one subset of these genes has revealed that they encode proteins required to produce an anti-tumor agent.

"We now have a new tool we can use to find secondary metabolites that are of pharmaceutical interest," says Keller. Although the team worked on a widely studied fungus, Aspergillus nidulans, the method can be used to find secondary metabolites in many other fungal species.

While primary metabolites are essential compounds that aid basic growth and reproduction in fungi, secondary metabolites are not required for life. "Secondary metabolites are bioactive compounds that are only produced at select times during the life cycle of the organism," explains Keller, noting that the compounds can help fungi survive various environmental stressors.

Some secondary metabolites have powerful heath effects in humans, such as penicillin, which certain fungi produce in the presence of bacteria. Fungi are also natural producers of antiviral agents, antifungals, other antibacterials, immunosuppressants and the popular cholesterol-lowering drug lovastatin.

Earlier methods to find secondary metabolites located just one compound at a time, and sometimes required prior knowledge about the compound of interest. Even after the genetic sequence of Aspergillus nidulans was completed in 2003, the search for secondary metabolites in that species continued. Although scientists were able to pinpoint the locations of genes believed to be involved in the production of secondary metabolites by pouring over the sequence data, the actual activity of the genes and their gene products still needed to be confirmed.

The method developed by Keller and her colleagues solves this problem and promises to broaden the search for medically useful agents by taking a powerful, genome-wide approach. Their technique is called "genomic mining" because of its robust ability to dig through an entire fungal genome to locate nearly all the hidden gems-the actively-produced secondary metabolites-at once.

The key to Keller's approach lies in a single fungal protein called LaeA. A few years ago, her team discovered that the presence of LaeA is required to turn on the genes that manufacture secondary metabolites in Aspergillus nidulans. "LaeA controls the production of secondary metabolites," says Keller.

For some as-yet unknown biological reason, all the genes required for the production of any given secondary metabolite-usually between three and two dozen genes-are located right next to each other along the chromosome in a gene cluster. These groups of genes stand out in certain scientific analyses, making secondary metabolite gene clusters easy to spot. Keller capitalized on these facts to find actively-produced secondary metabolites.

In one experiment, Jin Woo Bok, a research scientist in Keller's lab and co-author on the paper, deleted the LaeA gene in Aspergillus nidulans. Using a device called a microarray, the research team measured the activity of every gene in the LaeA-free mutant. "We looked at the entire 10,000 genes in this fungus," says Keller.

Keller's team searched the genome for groups of contiguous genes that were inactive in the mutant. They knew these were very likely to be gene clusters involved in the production of secondary metabolites.

In a parallel experiment, Bok designed a fungal strain that produced extra amounts of LaeA. This time, the team searched for groups of over-active genes, which they knew were likely secondary metabolite gene clusters.

The team's new approach located 40 active gene clusters; previously, fewer than ten secondary metabolites were known in this widely studied fungal species.

The newly discovered secondary metabolite gene clusters are already beginning to yield potentially useful medical agents. The team found an anti-tumor agent never before seen in Aspergillus nidulans. "It's just one of many [secondary metabolites to discover]," says Keller. "It's just the tip of the iceburg."

Thirty or so secondary metabolites await analysis in Aspergillus nidulans alone. Additionally, scientists suspect LaeA may play a similar role in all Aspergillus species, meaning there are over 180 fungi that can be mined using this technique. Keller also believes the mining strategy might work outside the Aspergillus genus with some modifications.

"This is really exciting," says Keller. "It's a method to find new metabolites, some of which we hope to be important ones."

In addition to Keller and Bok, other members of the research team include Jeremy D. Glasner at UW-Madison; Lori A. Maggio-Hall, formerly of Keller's lab and now at DuPont; Dirk Hoffmeister at the Pharmazeutische Biologie und Biotechnolgie in Freiburg, Germany; and Renato Murillo at Escuela de Quimica y CIPRONA in San Jose, Costa Rica. The work was supported financially by the National Science Foundation and the National Institutes of Health.